Huntington's Disease (HD) is an inherited autosomal dominant genetic disorder caused by expansions of CAG repeats (polyglutamine-polyQ) at the N-terminus, within exon 1, of the HD protein. HD is marked by neuronal tissue degeneration and appears be due to the development of protein aggregates that arise initially from the misfolding of the mutant HD protein. Recent studies suggest that mutant Htt can nucleate protein aggregation and interfere with a multitude of normal cellular functions. protein. Recent studies suggest that mutant Htt can nucleate protein aggregation and interfere with a multitude of normal cellular functions.
The extent of polyglutamine expansion is correlated with the severity of the symptoms and their onset while the pathology of the disease and neuronal cell death are thought to be associated with protein misfolding and protein aggregation. These aggregates are usually seen in the nucleus but can also be found in the cytoplasm. Protein aggregates develop via a complex biochemical process with intermediates being visible during the process. PolyQ tracts within the pathogenic range induce a protein insolubility whereas Htt with nonpathogenic length maintains a measured degree of solubility.
Consistent with the aggregate toxicity hypothesis, inhibition of aggregate formation has been shown to have beneficial effects on the progression of HD in the R6/2 mouse model. The implication of the polyQ aggregates in cytotoxicity validates them as targets for novel therapeutics. Despite the lack of details surrounding the molecular structure of the polyQ aggregates, high throughput screening for compounds that inhibit their formation have produced some promising results. Several compounds, including Congo Red and Clioquinol, have been reported to inhibit the aggregation process in the R6/2 mouse model but their neurotoxicity tempers enthusiasm. Thus, identifying molecules that show efficacy with minimal toxicity should be an important consideration in the search for HD therapeutics.
Synthetic ODNs (ODNs) provide a model category of reagents that meet some of these requirements. ODNs are synthetic polymers that are produced in highly purified quantities in a cost-effective way and the technology surrounding ODN synthesis has advanced dramatically in the last 10 years. Recently, Parekh-Olmedo et al. (J. Mol. Neurosci. 2004; 24(2):257-67) showed that certain classes of ODNs can inhibit aggregation. One of these groups is the G-rich ODN (GROs) class which have been used previously as aptamers to block protein function. Specifically, GROs have been shown to bind directly to STAT3 and interact with regions of the protein that enable dimerization and in another instance, GROs have been shown to block the integration of the HIV into the host chromosome by interacting with the HIV integrase. In both cases, the GRO forms a structure known as a G-quartet which arises from the association of four adjacent G-bases assembled into a cyclic conformation. These structures are stabilized by von Hoogstein hydrogen bonding and by base stacking interactions. These molecules exhibit a very compact structure which allows them to interact productively with functionally important protein domains.
Much of the focus on developing therapeutics that block aggregate formation comes from a wealth of data associating HD pathogenesis with the presence of cellular inclusion bodies. But, recent evidence from in vitro and in vivo studies suggest that Htt inclusions may not be toxic to the cell or lead to neuronal degeneration. In fact, Hayden and colleagues have created an exciting mouse model that shows no long term effect of Htt inclusions on behavior or viability. It may be true that inclusion bodies are neuroprotective and eliminating them may actually increase the potential for neurotoxicity.
Huntington's disease is caused by an increase in the length of the poly(Q) tract in the huntingtin (Htt) protein, which changes its solubility and induces aggregation. Aggregation occurs in two general phases, nucleation and elongation, and agents designed to block either phase are being considered as potential therapeutics.
Intracellular aggregates of Htt have long been considered phenotypic evidence of the neurodegenerative disorder Huntington's Disease. It is, however, not clear how the appearance of such inclusion bodies relates to the pathogenesis of the disease. A number of model systems have been designed to screen for therapeutic agents that can inhibit aggregation. Some of these assays measure the inhibition of fusion protein aggregation, proteins containing a fragment of Htt (here, GST-Q58-Htn) and a marker/reporter protein, often eGFP. The Htt component of this fusion protein harbors an expanded polyQ stretch.
As such, efforts to find a therapy for HD have focused on agents that disrupt or block the mutant Htt aggregation pathway.
It is well known in the art that G-rich DNA and RNA form inter- and intramolecular four-stranded structures known as G-quartets. G-quartets are formed when four G-bases are associated into a cyclic Hoogsten H-bonding arrangement wherein each G-base makes two H-bonds with its neighboring G-base. Ultimately, G-quartets stack on top of each other, giving rise to tetrad-helical structures. The stability of these G-quartets is related to several factors, including the presence of monovalent cations such as K+ and Na+, the concentration of G-rich ODNs present, and the sequence of the G-rich ODNs being used.
Many G-rich ODNs (GROs) have demonstrated significant cell-signaling factors. Identified GROs have been implicated in several cell functions and a variety of disorders. In particular, certain GROs display effective antiproliferative activity when added to cancer cell lines (Bates et al., Antiproliferative activity of G-rich ODNs correlates with protein binding, J. Biol. Chem., 274, 26369-26377 (1999); Xu et al., Inhibition of DNA replication and induction of S phase cell cycle arrest by G-rich ODNs, J. Biol. Chem., 276, 43221-43230 (2001); Dapic et al., Antiproliferative activity of G-quartet-forming ODNs with backbone and sugar modifications, Biochemistry, 41, 3676-3685 (2002)). Specifically, it has been reported that treatment of tumor cells with GROs inhibits cell cycle progression by interfering directly with DNA replication, as opposed to normal skin cells that exhibited minimal disruption of the cell cycle when treated with the same GROs (Xu et al.).